Project Summary/Abstract Drastic changes in lifestyle and dietary trends have triggered a rapid, worldwide epidemic of chronic metabolic diseases including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD/steatosis). The need for more effective treatments of T2D and metabolic diseases has never been more urgent. This proposal investigates the innovative hypothesis that T2D results from deregulated function of hepatic Argonaute 2 (Ago2), a main component of the RNA-induced silencing complex (RISC). Our novel preliminary data indicates hepatic Ago2 plays a central role in regulating mitochondrial function in the pathogenesis of T2D. In mammals, there are four Argonaute family proteins that play crucial roles in RNA silencing. Among them, Ago2 uniquely possesses endonuclease activity that is critical for the biogenesis of specific miRNAs and for mRNA cleavage. In the hepatic Ago2-deficient state, expressions of miRNAs, which are known to impair glucose metabolism, are selectively suppressed, whereas mRNAs enhancing mitochondrial function are increased. Intriguingly, our preliminary results identified that metformin treatment induces Ago2 functional changes associated with its endonuclease activity. These preliminary data suggest unique and pivotal roles for hepatic Ago2 in regulating glucose homeostasis, leading us to hypothesize that Ago2 regulates glucose and lipid metabolism in the liver through miRNA biogenesis and RNA silencing that affects mitochondrial function. Further, we hypothesize that increased Ago2-mediated RNA silencing contribute to hepatic steatosis and insulin resistance, and decreased Ago2 function mediates the therapeutic effect of metformin. Studies in this proposal will: (1) determine the Ago2 endonuclease activity important in T2D pathogenesis; (2) dissect Ago2 function in the therapeutic effect of metformin for improved glucose metabolism in T2D; (3) delineate Ago2-dependent mRNA silencing critical in glycemic control in T2D and in metformin's action. The goal of this application is to gain a clear mechanistic understanding of the role of the hepatic Ago2- miRNA axis in the pathogenesis and in the treatment of T2D. The long-term goal of this program is to identify novel preventive and therapeutic strategies for T2D.